Antibiotic having parasiticidal activity

ABSTRACT

1. AN ANTIBIOTIC ASPICULAMYCIN HAING THE STRUCTURAL FORMULA   1-(3,4-DI(HO-),5-(H3C-NH-CH2-CO-NH-CH(-CH2-OH)-CO-NH-   CH(-CH2-OH)-CO-NH-),6-(H2N-CO-)-TETRAHYDROPYR-2-YL-),   2-(O=),4-(H2N-)-1,2-DIHYDROPYRIMIDINE

Nov. H9, 1974 MAMORU ARA! ET AL 3,849,398

HAVING PARASITICIDAL ACTIVITY ANTIBIOTIC Filed Aug. 2, 1972 l l l l 3560 5000 zboo I800 1600 14110 1200 1600 860 United States Patent 3,849,398 ANTIBIOTHC HAVING PARASITICIDAL ACTIVITY Mamoru Arai, Tatsuo Haneishi, Hisashi Kayamori, Yoo

Patented Nov. 19, 1974 ice (2) Results obtained in the culture of strain No. 1040 on various media (observation was made after a twoweek cultivation at 27 C. in each medium, unless otherwise specified) are shown in Table 1. Generally, the aerial hypha and spores are powdery, and the mycelium $32 5 ig gf gggg i g zg gg figg 5333: 33 223 5 are small in number. Colors were determined according Filed Aug 2, 1972 sen 277,588 to Guide to Color IStandard (a manual published by Claims priority, application Japan, Aug. 3, 1971, PP Shlklsal y 46/523,536 (3) Physiological properties of strain No. 1040 are Int. Cl. C07d 51/52 shown in Table 2. US. Cl. 260211.5 AB 1 Claim 10 ABSTRACT OF THE DISCLOSURE TABLE 2 An antibiotic aspiculamycin having the structural formula Temperature range for growth l040 C.

Gelatin liquefaction moderate. NHZ Hydrolysis of starch moderate. Coagulation of skim milk at 25 and 37 C. I Peptonization of skim milk at and 37 C. i (pH 6.6). 0 N Melanin formation: HZNO On tyrosine-agar medium 0 On peptone-yeast iron agar 7 medium HOCH CHOCHN Nitrate reduction 0H WeCHZOH NHCCHZNHCH3 102i if iifiliaii iifliiil f ifi ifiiiiifii lii im fii The antibiotic has parasiticidal action against parasites T bl 3, on animals, such as pinworms and the like.

This invention relates to a novel antibiotic named as- TABLE 3 picularnycin.

We have found that the antibiotic aspiculamycin is sigggg 1- produced in a culture of Streptomyces toyocaensis var. D glucose aspiculamyceticus strain No. 1040, a novel actinomycetes, D fructose which was first isolated from a sample of soil collected 40 Sucrose in Hiraizurni, IWate Prefecture, Japan. The present anti- Inositol u biotic is isolated by extraction from the culture filtrate Raffinos and purified. It is markedly characterized in that the anti- 6 biotic has parasiticidal action against parasites on ani Lrhamnose n mals, such as pin Worm and the like, though it shows Cellulose a weak antibacterial activity against gram-positive, gramnegative bacteria and tubercule bacillus, etc.

Morphological characteristics of the aforesaid strain N 10 40 are as f ll In the search for known strains having properties simi- When observed under a microscope aerial Inycel- 131 to those Of strain N0. there may be mentioned ium extends from well-branched substrate mycelium, and slrepio'myces foyocaellsis WhiCh has been descrlbed in The at the top of the aerial mycelium a chain of spores is Actlnomycfles, 2 (Waksman, From the fact f d that strain No. 1040 produces simultaneously an anti- The aerial mycelium for-ms numerous cluster-like blOtlC, toyocamycin, in addition a 116W antibiotic aspiculabranches. The aerial hyphae are terminated with spirals 5 mycin in the Same manner as in Streptomyces loyomensis, which are in coils of 5 to 20 turns. Spore chains are it is considered that strain No. 1040 is a member of the generally in 50 or more spores per spore chain. Spores genus Streptomyces belonging to Streptomyces toyo- TABLE 1 -Mycelium s 1 M Medium Growth Aerial Substrate Reverse pigit ier it;

Sucrose nitrate agar Moderate Pale brown Colorless". Pale brown Non Glucose asparagine agar. do Brownish white Yellowish grey Do. iiifi iitii i fiif f? do i332 Tyrosine agar D0. Nutrient agar... n... o Do. Yeast-malt agar- Abundant- Pale yellowish brown do Do. Oatmeal agar d0 Brownish grey Yellowish grey Yellowish grey D0.

are spherical to elliptical in shape and 0.4-0.6 x 0.7-1.0 in size.

caensis. Strain No. 1040, however, differs from Streptomyces toyocaensis in the following respects.

Q) Strain No. 1040 forms a long spiral filament, whereas Streptomyces toyocaensis forms a short spiral.

( Strain No. 1040 forms a spiral filament on a starch/ inorganic salt medium, whereas Streptomyces toyocaensis does not form the same.

@ On any media, strain No. 1040 grows very well, compared with Streptomyces toyocaensis.

@ The two strains are somewhat different from each other in color of aerial and substrate mycelium when grown on various media.

@ Strain No. 1040 produces a new antibiotic, aspiculamycin.

From the above, strain No. 1040 has been identified as one of variants of Streptomyces toyocaensis and named Streptomyces toyocaensis var. aspiculamyceticus. Strain No. 1040 has been deposited under accession No. 1036 with Technical Research Institute of Microbial Industry, Agency of Industrial Science & Technology, Japan.

Having thus explained the characteristics of strain No. 1040, it is well known that various properties of Streptomyces are not definite, but may be easily varied naturally and artificially. Consequently, the strains usable in the present invention include all of the strains which belong to genus Streptomyces and which are capable of producing aspicularnycin.

Cultivation of the strain in the present invention may be carried out according to the method generally employed for Streptomyces. In carrying the cultivation into practice, advantageous results are obtained when the strain is inoculated into a liquid medium,- with approximately neutral pH and cultured at 25-35 C. under aerated agitation. The medium may contain, as a carbon source, starch, glucose, glycerine, etc., as a nitrogen source, meat extract, peptone, corn steep liquor, soybean meal, yeast, cotton seed meal, etc., and as an inorganic salt, sodium chloride, potassium chloride, calcium carbonate, phosphates or the like. Usually, the amount of aspicularnycin produced in a cultured broth reaches maximum in 40-120 hours.

To recover aspicularnycin from the cultured broth, any means commonly used in recovering natural prod ucts may suitably be employed. For instance, such means as relying on utilization of the dilference in solubility and in adsorbability on an ion exchange resin between aspiculamycin and the imprities, may be used alone or in combination or repetition.

For example, aspicularnycin may be recovered by filtration of the cultured broth after adjustment of its pH to acidic to neutral together with such filter aids as diatomaceous earth, removing mycelium, passing the filtrate onto a cation exchange thereby to have the present antibiotic adsorbed thereon and then eluting the adsorbed antibiotic with an appropriate acid, alkali or inorganic salt solution. In that case, two antifungal antibiotics, i.e. tetraene and toyocamycin, may be simultaneously separated from aspicularnycin, because the tetraene is not adsorbed onto the cation exchange resin and the toyocamycin is eluted at a pH lower than that of aspicularnycin. Aspiculamycin can also be recovered by adsorbing the culture filtrate onto activated carbon at an acidic to neutral pH and extracting aspicularnycin with an acidified aqueous methanol or aqueous acetone. The concentrated culture liquid may be further purified using a cation exchange resin and an anion exchange resin. After concentrating an aqueous solution containing the extracted, isolated and purified aspicularnycin, the solution is subjected to lyophilization or precipitation with an organic solvent, e.g. methanol, ethanol, acetone, etc., lwhereby aspicularnycin can be obtained in the form of a white powder. Aspiculamycin is crystallized from aqueous acetone.

The new antibiotic of the present invention which is named aspi'culamycin has the following structural formula.

II NOC OH HOCH -CIIOC'HN NH-COWCH(CHZOH)NH-CO41124111143013 The physico-chemical properties of aspicularnycin are given below.

(1) White needle crystals.

(2) Melting point: Decomposition begins gradually at 200 C. or above, though an accurate melting point is undeterminable.

(3) Elementary analysis for C H O N /2H O: Calculated: C, 42.37%; H, 5.58%; N, 20.82%; 0, 31.22%. Found: C, 42.63%; H, 5.78%; N, 20.52%; 0, 31.07%.

(4) Molecular weight: 490.5, as measured according to the vapor pressure method; 530, as calculated on the basis of the molecular formula.

(5) Specific rotation: [a] =+54.0 (C.=1, Water).

(6) Ultraviolet absorption spectrum: As shown in FIG. 1, values of a maximum absorption in water and 0.05 N NaOH solution are 236 m and 268 my.

( lfin;

respectively, and the value in 0.05N HCl solution is 276 m (7) Infrared absorption spectrum: The spectrum as measured in Nujol mull is as shown in FIG. 2. The major absorption occurs at 3350, 3200, 1690, 1660, 1610, 1530, 1495, 1290, 1210, 1090, 1030, 940, 850, 790 cmr (8) Solubility: Aspiculamycin is easily soluble in water, but insoluble in methanol, ethanol, acetone, chloroform, etc.

(9) Color reaction: Positive to Ninhydrin and 2,4-dinitrophenyl hydrazine reactions. Negative to Molisch, Anthrone, Fehling reactions, and silver nitrate is not reduced.

(10) A basic substance with pKa' 3.90zpKa 8.24.

(11) Chromatography: By paper chromatography when developed with Water and water-saturated butanol using Toyo Filter Paper No. 51, Rf values are 0.37 and 0, respectively. By a thin layer chromatography on Eastman chromagram Sheet 6065, R value in a solvent system constituting of a mixture of n-butanol, acetic acid and water (4:1:1) is 0.05, and 0.40 in a mixture of n-propanol, pyridine, acetic acid and water (15 :1023210).

The biological activities of aspicularnycin are set forth below:

(1) Table 4 shows the minimal inhibitory concentration of aspicularnycin against various bacteria, fungi, yeasts and plant pathogenic microorganisms. The activities against bacteria 'were determined on a nutrient agar after incubation at 37 C. for 24 hours. In the case of tubercle bacillus, the determination was conducted after 48 hour incubation at 37 C. for 24 hours. In the case of tuburcule and yeasts on Sabourauds agar and plant pathogenic fungi on a potato-sucrose agar were incub ed at 25 C- for 48 hours, respectively.

TABLE 4 Minimal inhibitory (2) When acute toxicity of the present antibiotic in mice by intravenous injection were examined, three-fifths theregii zgiz g dwells 209p fi fi i fi fifi l z of were killed at a dose of 25 mg./kg., and three-fifths Staphylococcus aureus 5 5 h of s rvived a a dose of mg./kg. In the case of oral Staphylococcus aureus 1557 400 administration, two-fifths of the mice were killed at a dose Staphylococcus aureus 193 400 of 125 mg./kg. gif 2 (3) Aspicularnycin has remarkable activity to inhibit Sam-"a [urea 200 10 parasites on animals from ovulation as well as parasiticidal Corynebacterium xerosis 400 action. For example, Table 5 shows the results obtained in Aeromonas punclata 400 the ovulation inhibition test on pin Worms, such as Pseudomonas {emginosa 400 Syphacia obvelate and Aspiouluris tetraptera in mice. Mice Proteus vulgarts 40 0 Escherichia Cali NIH] 200 taied on a basal diet were divided mto three groups, Escherichia Coll- 200 each consisting of 5 mice: group 1 was injected With basal Escherichia coli (resistant to streptothricin) 200 diet Without p amycin; groups 2 and 3 were injected Escherichia (resistant to Streptomycin) 100 with basal diet with 100 mg. and 50 mg. of aspiculamycin Escher ch a colt (res stant to kanamyc1n) 50 per 1 kg. of the basal diet respectively. Escherzchza colz (resistant to chloramphemcol,

tetracycline) 50 This test 1s intended to observe the progress of ovula- Escherichia coli 97 (multiesistant) 400 tion with a lapse of time of 4 Weeks after administration, Mycobacterium smegmatis 607 50 and, at the same time, to examine the parasiticidal efiect g gilbicans W by counting the number of parasites in the intestine at cc taro ces cerevzsza 2 Trichophytzn imerdl-gimele 0 autopsy after completlon 0f the test- I cryptococcus neoformans 400 As can be seen from the results shown in Table 5, perfect Aspergillus nigar 400 inhibition of ovluation is attained within 4 Weeks in both Penicillium clzrysogenum 400 cases of the administration with dosages of 100 mg. and Hornwaienimm pedmmi mg./kg., and from the results obtained in the autopsy, it is recognized that a perfect parasiticidal effect is attained.

Gloeosporium kaki 400 TABLE 5 Group 1 Group 2 Group 3 Day on which administration +t t +1 +t +r i t++t +t t t t;; t::::::t::iti:

autopsy y 6 7 9 0 6 0 0 0 0 0 0 0 0 0 Furthermore minimal iphibitory conceptrations on M Thus, it has been confirmed that aspiculamycin is a new coplasma of asplculamycm are shown In the following antibiotic in view of the fact that no antibiotics having such table" physicochemical and biological properties as mentioned Mycoplasma: M.I.C. g/ml.) above have been heretofore known.

Mycoplasma mycoides var. mycoides 25 The present invention is illustrated below with reference M. agalactiae 1.56 5 to an example. It is evident, however, that the objects of M. mycoides var. capri 1.56 the invention may be accomplished even by employing M. arthritidis 6.25 many means which have not illustrated concretely in the M. laidlawii 25 specification but are within the scope of the present inven- M. bovigenitalium 6.25 tion, since various properties of aspiculamycin have al- M. pulmonis 1.56 ready been unveiled hereinabove.

M. gailisepticum 1.56 EXAMPLE M. canis 12.5

M. felis 3.12 A liquid medium containing 3.0% starch, 1.0% meat M. hyorhinis 0.78 extract, 1.5% pharmamedia and 2.0% corn steep liquor M. hominis type 1 3.12 is adjusted to pH 7.6 and the medium is sterilized at C. for 30 minutes. The medium is inoculated with a strain of Streptomyces toyocaensis var. aspiculamycetious, and the strain is cultured under aerated agitation. The cultivation is continued while monitoring the potency of a cultured broth using Escherichia coli NIH] as a test microorganism until the maximum potency is attained. Usually, the potency reached the highest level in to 6 days when 100 ml. of the medium placed in a 500 ml. Sakaguchi flask was subjected to shaking culture. Furthermore, when the culture broth which had been precultered for 48 hours was inoculated into 60 l. of the medium in 100 l. fermentors and the cultivation was carried out with agitation at a rate of 150 r.p.m., while passing therethrough sterile air at a rate of 60 l./min., production of aspiculamycin reached the highest level in about 90 hours. In a filtrate of the final culture broth thus obtained, there were simultaneously produced two antibiotics, a tetraene-like antibiotic showing antibacterial activity against gram positive bacteria and yeasts, and toyocamycin.

Extraction and purification of aspiculamycin produced are effected according to the following procedure.

(1) To 5 l. of the culture broth obtained in the Sakaguchis flask according to the procedure as mentioned above, there was added of diatomaceous earth, and the mixture was filtered. The resulting filtrate to which 2% of an activated carbon had been added was agitated and then filtered. The activated carbon was washed with 2 l. of water. The activated carbon thus washed was extracted 2 times with 1 l. of 50% acetone acidified with hydrochloric acid to pH 2. After neutralization, the resulting extract was concentrated under reduced pressure to obtain 500 ml. of a concentrate containing aspiculamycin together with toyocamycin. Aspiculamycin present in the concentrate thus obtained was further purified by column chromatography on Amberlite IRC-SO which is a weakly acidic cation exchange resin. The concentrate Was added onto Amberlite IRC50 of NH type, whereby toyocamycin which had been mixed therein was recovered in a non-adsorbed portion to be removed. After waterwashing, elution with 0.5 N NH OH was carried out. Active fractions were concentrated and, after removal of ammonia, impurities were removed by adsorption onto Dowex 1 X l of OH type which is a strongly basic anion exchange resin, the active fractions passing therethrough were concentrated and lyophilized to obtain 0.8 g. of aspiculamycin in the form of a white powder.

(2) Sixty (60) liters of the aforesaid tank cultured broth was filtered together with 6 kg. of diatomaceous earth. The filtrate was passed through a resin column packed with 6 1. of Amberlite IRC-SO of H+ type, thereby removing tetraene, and the adsorbed fractions were eluted with 1N NH OH. Toyocamycin was first eluted and then aspiculamycin was eluted. Fractions containing the aspiculamycin were concentrated, and 2 l. of the concentrate was adsorbed onto Amberlite CG50, which had previously been equilibrated with ammonium sulfate buffer solution of pH 7.0, and eluted with 0.05 M diammonium hydrogen phosphate. Active fractions containing aspiculamycin were adsorbed onto Amberlite CG-50 of H+ type, washed with water, eluted with 0.5N NH OH, desalted and then concentrated. The concentrate thus obtained was precipitated with acetone, and the precipitate Was collected by centrifugal separation. The collected precipitate was dissolved in water and lyophilized to obtain 2.5 g. of aspiculamycin in the form of a white powder. One gram of powder thus obtained was dissolved in 10 ml. of water and 40 ml. of acetone was added thereto, thereby crystallizing aspiculamycin. The yield of crystalline antibiotic was 200 mg.

In the accompanying drawing, FIG. 1 shows an ultra violet absorption spectrum of aspiculamycin, and FIG. 2 shows an infrared absorption spectrum.

What we claim is:

1. An antibiotic aspiculamycin having the structural formula on aoca cuocau NHCOCIKCH2OH)NHC0CH2NHCH3 References Cited UNITED STATES PATENTS 3,625,940 12/1971 Suzukietal 260211.5 R

JOHNNIE R. BROWN, Primary Examiner US. Cl. X.R.

l80; 2602l 1.5 R; 424- 

1. AN ANTIBIOTIC ASPICULAMYCIN HAING THE STRUCTURAL FORMULA 